This invention relates generally to the enrichment of one or more desired populations or subpopulations from a sample having a plurality of cell populations to obtain the desired populations or subpopulations alone or the enriched populations or subpopulations with other populations, but with one or more undesired subpopulations removed therefrom. More particularly, the invention is directed to enriching the desired population or subpopulation such as cells from bone marrow, vertebral body marrow, or blood, by binding undesired populations or subpopulations to relatively dense particles and utilizing gravity sedimentation to separate the undesired populations or subpopulations from the remaining sample supernatant containing the enriched populations or subpopulations.
The enhancement or enrichment of a population or a subpopulation of a sample can be utilized for many types of applications. The pluripotent hematopoietic stem cell (HSC) population, which originates from the bone marrow, gives rise to all the red blood cells (RBC's) and white blood cells (WBC's) of the human body. These cells constitute a low percentage of the bone marrow (1-3%) and peripheral blood (&lt;1%), and as for any rare event cell type, it is difficult to study the HSC population without first enriching for the population. Both experimentally and clinically, it has become highly desirable to enrich for and/or isolate the HSC population, which population is identified via cell surface expression of the CD34 antigen. The HSC population is also referred to hereinafter as the CD34 population.
The potential applications for the HSC population include, but are not limited to, bone marrow transplantation for: treatment of hematologic malignancies, such as leukemia and lymphoma; treatment of solid tumors, such as breast and ovarian cancer; enhancement of graft acceptance and induction of donor specific tolerance in recipients of solid organ or cellular transplants; amelioration or cure of autoimmune syndromes, such as insulin dependent diabetes mellitus and systemic lupus erythematosus; and cure of inborn errors of metabolism. In addition, purified stem cells are required for many gene therapy protocols, which may eventually be utilized to treat all of the above conditions. The ability to isolate the HSC population will also allow for the expansion of the pluripotent HSC population and desired subpopulations of the HSC population, such as precursor dendritic cells, which can subsequently be utilized in all of the above applications. With the advent of techniques which allow for successful bone marrow transplantation between genetically disparate individuals, isolation of the HSC population from the peripheral blood or bone marrow of normal volunteers, followed by expansion, cryopreservation, and banking of the cells for utilization in various clinical protocols will soon be possible. Isolation and cryopreservation, of the HSC population from human cord blood at birth can allow for preservation of that individual's cells which then can be employed for the treatment of multiple disorders should they arise in that individual's lifetime.
Many prior art techniques have been developed for the enrichment/isolation of the HSC population, all of which require removal of mature RBC's, which can interfere with freezing and thawing of marrow cells and which can also interfere with certain chemical purging techniques, utilized to eliminate tumor cells from the marrow inoculum. The vast majority of the prior art HSC population enrichment protocols involve the use of density gradient centrifugation over ficoll-paque or other gradient materials, in order to enrich for mononuclear cells and eliminate mature RBC's. Alternative prior art techniques of obtaining mononuclear cell preparations include utilization of specialized blood cell processors which result in concentration of the mononuclear white blood cells. Furthermore, after enrichment for mononuclear cells and removal of mature RBC's, the total percentage of CD34 positive HSC in the processed population is still only on the order of 1-3%, and further processing must be undertaken in order to enrich for the HSC population. Both positive and negative selection techniques have been employed to enrich for the HSC population. Positive selection for the HSC population has been accomplished by exploiting CD34 specific monoclonal antibodies, avidin and biotinylated anti-CD34, or anti-mouse immunoglobin specific polyclonal antibody and murine anti-CD34 (bound to flasks, columns, particles/beads, or some other substrate) to pull out the HSC population. Negative selection techniques employ monoclonal antibodies which can bind to lineage specific markers, not present on the HSC population, to specifically remove the non-HSC population. All of these procedures are time consuming, unwieldy, and costly. In addition, due to the extensive manipulation of the HSC population, cell recovery of the desired HSC population is low.
Currently, following gradient processing, several prior art enrichment approaches involve the utilization of a plurality of magnetic microspheres particles/beads, typically formed of a polymer based magnetic material of a relatively low density. Unlike the present invention, described hereinafter, these microspheres are selected to be of a relatively low density, because the microspheres are mixed with the bone marrow or blood and specifically are designed not to settle out by gravity sedimentation. The microspheres are typically of a small size, generally about or less than one micron in diameter. However, one product sold by Dynal, Inc. of Great Neck, N.Y., utilizes magnetic polymeric microspheres having a nominal diameter of 2.8 or 4.5 microns with a low microsphere density on the order of 1.5 gm/cc. The prior art magnetic microspheres are intended to be maintained in suspension in the sample and consequently are designed for very slow or substantial elimination of gravity settling in the sample suspension.
The magnetic microspheres have at least one antibody bound thereto specific to the population or subpopulation desired to be removed. Often, such as in the Dynal process, a first monoclonal antibody is bound to the cells of interest and a second antibody specific to the first monoclonal antibody is bound to the microspheres. The cells typically are isolated from whole blood or bone marrow and then washed prior to binding the monoclonal antibody thereto, which washing step causes a non-discriminant loss of cells including those desired to be enriched. The microspheres and cells then are mixed together to bind the microspheres to the cells via the first and second antibodies. For removing cell populations from blood or bone marrow to enrich the desired population, a sample generally requires preprocessing by density gradients or RBC lysis and then would be mixed with a plurality of the antibody bound microspheres and then placed in a magnetic field. The remaining sample or supernatant containing the population to be enriched is removed while the microspheres are held in the magnetic field.
The magnetic removal procedure presents several problems. The procedure also removes a number of cells non-specifically from other populations during each removal step. The non-specific removal of cells can become more of a problem when a large sample volume is being utilized, such as five (5) ml and larger, which volume then requires a large number of the magnetic microspheres. When the magnetic microspheres then are placed in a magnetic field, non-specific trapping and removal of other non-targeted cells often occurs. This decreases the yield, i.e., the percent of the desired population remaining which is to be enriched. A single removal step results in a varying yield of a relatively low percent with each succeeding removal step, if utilized, also reducing the yield of the population to be enriched. Further, the magnetic microspheres are relatively expensive.
Other methods of positive selection, including antibody labeled surfaces, have been utilized for selecting and hence enriching populations or subpopulations of cells from a mixture of different cell types. These methods usually have antibody covalently attached to a plastic surface or to polymer particles in a column. In general, the mixed cell population is combined with the attached antibody, either by adding them to a column and letting them incubate or by letting them settle onto a surface. These procedures work optimally when the red blood cells (RBC's) and plasma have been initially removed from the mixed cell population by preparation of either a buffy coat or a mononuclear population, obtained by density gradients, followed by washing the cells and combining them with the antibody labeled surface. Both methods also require preparation of the separation system and washing with a buffer prior to use, which with incubation times of thirty to sixty (30-60) minutes with the antibody, results in a procedure which takes a minimum of three hours for the column and flask method. These methods can be used for positive selection for the cell population of interest.
The method and apparatus embodying the invention can be utilized with a variety of immunological reactions, such as immunological reactions involving reactants and cells. Cells are the least structural aggregate of living matter capable of functioning as an independent unit. For example, cells can be human RBC's, WBC's, including the HSC population, cancer or other abnormal cells from tissue, bone marrow, VBM and/or from blood samples from human or animal sources.
As utilized herein, the term "reactant" defines various molecule(s), such as monoclonal or polyclonal antibodies, which detect and react with one or more specific complementary molecule(s), such as antigens, which are on the surface of a cell. Some examples are given below:
______________________________________ Reactant Specific Molecule ______________________________________ Antibody Antigen Drug Drug Receptor Hormone Hormone Receptor Growth Factor Growth Factor Receptor Lectin Carbohydrate Molecule Enzyme Cofactor or Inhibitor ______________________________________
The reactants couple or bind to the specific molecule(s) on the cells.
It would be desirable to have an effective method of removing one or more populations or subpopulations without effecting the remaining population or populations to be enriched in a sample, such as whole blood, bone marrow or VBM. The technique should obviate the need for gradient centrifugation, specialized cell processors or lysis procedures. The method should be inexpensive, fast, result in a high yield of the population to be enriched and not be restricted in the volume of sample to be acted upon.